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Related Experiment Video

Updated: Jun 22, 2026

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

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Published on: November 12, 2020

Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture.

Marcel Leutenegger, Michael Gösch, Alexandre Perentes

|June 9, 2009

Summary

Researchers developed a new method using gold apertures to significantly reduce the sampling volume for fluorescence fluctuation spectroscopy (FFS). This breakthrough enhances single-molecule dynamics observation in biological samples with high fluorophore concentrations.

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Last Updated: Jun 22, 2026

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

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Published on: November 12, 2020

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Area of Science:

Biophysics
Spectroscopy
Nanotechnology

Background:

Observing single molecule dynamics using fluorescence fluctuation spectroscopy (FFS) requires very low fluorophore concentrations.
Micromolar concentrations common in biological samples challenge conventional FFS methods relying on confocal instrumentation.

Purpose of the Study:

To investigate enhanced confinement of the sampling volume for fluorescence fluctuation spectroscopy.
To overcome limitations of conventional FFS in biological samples with high fluorophore concentrations.

Main Methods:

Utilized sub-wavelength sized apertures in a thin gold film to create a highly confined near-field.
Characterized the gold apertures using fluorescence correlation spectroscopy (FCS).
Measured the effective sampling volume compared to confocal instrumentation.

Main Results:

Demonstrated light confinement beyond the far-field diffraction limit using gold apertures.
Achieved an order of magnitude reduction in the effective sampling volume compared to confocal microscopy.
Successfully characterized the enhanced confinement with fluorescence correlation spectroscopy.

Conclusions:

Near-field confinement via sub-wavelength gold apertures significantly reduces the effective sampling volume for FFS.
This nanophotonic approach offers a promising solution for observing single molecule dynamics in high-concentration biological systems.
The method enhances the sensitivity and applicability of fluorescence fluctuation spectroscopy in biological research.